Nucleus downscaling in mouse embryos is regulated by cooperative developmental and geometric programs

Abstract

Maintaining appropriate nucleus size is important for cell health, but the mechanisms by which this is achieved are poorly understood. Controlling nucleus size is a particular challenge in early development, where the nucleus must downscale in size with progressive reductive cell divisions. Here we use live and fixed imaging, micromanipulation approaches, and small molecule analyses during preimplantation mouse development to probe the mechanisms by which nucleus size is determined. We find a close correlation between cell and nuclear size at any given developmental stage, and show that experimental cytoplasmic reduction can alter nuclear size, together indicating that cell size helps dictate nuclear proportions. Additionally, however, by creating embryos with over-sized blastomeres we present evidence of a developmental program that drives nuclear downscaling independently of cell size. We show that this developmental program does not correspond with nuclear import rates, but provide evidence that PKC activity may contribute to this mechanism. We propose a model in which nuclear size regulation during early development is a multi-mode process wherein nucleus size is set by cytoplasmic factors, and fine-tuned on a cell-by-cell basis according to cell size.

Figure 1. Nuclear volume and N/C ratio during early embryo development.

(A) Representative images of embryos used to measure volume. (B) Quantification of nucleus size, related to cell; size, on a cell by cell basis. Each data point represents an individual cell, colour codes reflect developmental stage. (C) ‘Zoomed’ representation of 8-cell embryo data. (D) Data expressed as mean nuclear/cytoplasmic ratio. Note that the N/C ratio progressively increases through preimplantation development. 12, 28, 48, 86, 88 and 82 nuclei analysed at 1,2,4,8-cell, morula and blastocyst stage respectively, over the course of 2 replicates.

Figure 2. Nuclear downscaling occurs independently of the first cell fate decision.

Cells were treated with nominally Ca²⁺-free media for <10 seconds to cause inter-cellular adherence to be partially lost, causing cells to round. Embryos were then fixed and nuclear volume and N/C ratio analysed in Oct4-positive cells, using the same methods as previously. Five cells of each cell type were analysed in 10 blastocysts, across two experimental days.

Figure 3. Experimental cytoplasmic reduction to probe the role of cell size in nucleus-size setting.

(A) Experimental design. (B) Examples of control and cytoplasm-reduced embryos fixed and labelled at 4-cell stage. (C) Analysis of nuclear and cell volume in the resulting 4-cell embryos. Each point represents one cell. Red manipulated, white controls. Note the tight relationship between cell and nuclear size. R² = 0.67. Data from 82 nuclei over 4 replicates.

Figure 4. Evidence that N/C ratio is developmentally regulated in mouse.

(A) Analysis of nucleus size in binucleated embryos. Experimental plan is shown to the left, and an example of a binucleated embryo in the centre panel. Data to the right compares nucleus size in binucleated embryos with normal 4-cell controls. 40 control and binucleated embryos each were examined over 2 days. (B) Analysis of nucleus size in ‘double-sized’ blastomeres. Note that the resulting 8-cell-stage nuclei in ’double sized’ blastomeres (n = 42) have nuclei the same size as control 8-cell embryos (n = 56), which are substantially smaller than control 4-cell embryos (n = 40). Data collected over 3 replicates. Different letters indicate P < 0.01 ANOVA (actual values stated in the main text).

Figure 5. Evidence that PKC, but not nuclear import, contributes to nucleus size-setting.

(A) NLS::GFP FRAP in 2-cell, 4-cell, and 8-cell stage embryos (n = 10, 9, 9 respectively from 3 replicates). Note there is no difference in the rate of recovery between developmental stages. (B) Live imaging of CAAX::GFP and H2B::RFP expressing embryos after interphase addition of PMA or chelyrethrine. (Chel, n = 20, PMA, n = 14 from 2 replicates) (C) Analysis of the effect of PMA or chel upon nucleus size following NER. A cartoon is shown of the experimental design, and images show typical examples of each treatment. Note that the treatments cause reciprocal changes in nucleus size. A minimum 37 measurements per experimental group from three replicates. All groups are significantly different to each other within a given developmental stage (P < 0.01 ANOVA for each comparison, actual values stated in main text).